Although toxins associated with major bacterial diseases are known to modify cytosolic substrates within mammalian cells, we still do not have a clear understanding of how any toxin transfers its enzymic moiety across a membrane. Our primary aim will be to pursue this goal using anthrax toxin (AT) and diphtheria toxin (DT) as systems for investigation. AT and DT have in common a low endosomal pH that induces their B moieties to form aqueous pores (channels) in membranes as an essential step in translocation. These toxins are structurally unrelated, however, and use different strategies of pore formation. Protective Antigen (PA), the B moiety of AT, forms ring-shaped heptamers at the mammalian cell surface, which undergo a conformational change under acidic conditions of the endosome to generate a transmembrane 14-strand - barrel. This insertion into the endosomal membrane mediates translocation of noncovalently bound Edema and Lethal Factors (EF and LF), the enzymic A moieties of AT, to the cytosol. In contrast, DT is a single- chain toxin that contains an alpha-helical pore-forming domain (T-domain), which inserts into bilayers and forms a channel under acidic conditions. Channel formation in model membranes is accompanied by translocation of the N-terminus of T-domain and covalently tethered A chain. With these models, the crystallographic structures of the native proteins, and physiological data on toxin action we will probe the fundamental mechanisms of translocation in these two systems. Questions to be investigated include: What are the structures of the isolated PA pore; EF or LF complexes of the pore or prepore; the PA receptor (ATR) complexed with monomeric or heptomeric PA; and finally the DT channel? What conformational changes occur in domain 2 of PA, the channel forming domain, as the prepore converts to the pore? Can one devise model membrane systems to study translocation by AT in vitro, similar to those used for DT? Do LF and EF pass through the lumen of the pore, through adjacent lipid regions, or through the protein:lipid interface as they transit the membrane? What is their conformation during membrane transit? These and other unanswered questions relating to toxin action will yield insights into mechanisms of bacterial pathogenesis and will contribute to understanding how macromolecules insert into and cross membranes.